There is compelling evidence for the involvement of presynaptic inhibition in controlling normal synaptic transmission and preventing excessive neurotransmitter release at mammalian central synapses. The presynaptic site is an effective target for modulation of synaptic transmission and presynaptic voltage-dependent calcium channels play a significant role in controlling transmitter release. To address our long-term objective of understanding the basic presynaptic mechanisms underlying modulation of synaptic transmission in mammalian central synapses, we propose to investigate the direct and indirect role of presynaptic calcium during inhibition of synaptic transmission at hippocampal CA3/CA1, MF/CA3 and PP/GC excitatory synapses. This in vitro study will employ hippocampal brain slices and optical imaging techniques. We will selectively load presynaptic terminals with ion-sensitive indicators to investigate resting levels and transients of presynaptic calcium and presynaptic potassium. In addition, we will use voltage- sensitive dyes to measure presynaptic action potentials. Specific blockers will be utilized to identify and quantify the types of presynaptic voltage-dependent calcium channels involved in synaptic transmission. We will study presynaptic calcium during the application of neuromodulators with presumed inhibitory presynaptic action and identify the types of calcium channels involved and their quantitative inhibition. Through the use of advanced optical techniques, the proposed studies will provide new and important insight into the presynaptic modulation of mammalian synaptic transmission. This insight will contribute to the understanding of normal and pathological synaptic transmission. Excessive release of excitatory neurotransmitter has been observed during episodes of epilepsy and after brain damage. Control of this release by presynaptically acting endogenous neuromodulators could be the basis of future therapeutic interventions. We will address the following specific aims: 1) To discriminate the types of presynaptic VDCCs at principal hippocampal excitatory synapses. 2) To investigate resting levels and influx of presynaptic Ca2+ during presynaptic inhibition of evoked synaptic transmission.